1. Volume 113, Issue 11, Pages 2496-2507 (December 2017)
SOCE Is Important for Maintaining Sarcoplasmic Calcium Content and Release in Skeletal Muscle Fibers 
Mónika Sztretye, Nikolett Geyer, János Vincze, Dána Al-Gaadi, Tamás Oláh, Péter Szentesi, Gréta Kis, Miklós Antal, Ildikó Balatoni, László Csernoch, Beatrix Dienes 
Biophysical Journal 
Volume 113, Issue 11, Pages (December 2017)
DOI: /j.bpj
Copyright © 2017 Biophysical Society Terms and Conditions

2. Figure 1
Reduced SOCE in Cmpt mice. (A and C) Representative xy images showing the response to various solution exchanges and the consequent SOCE activation in enzymatically isolated FDB fibers of C57/BL6 WT (A) and Cmpt (C) mice. (B and D) During manual solution exchanges, the fluorescence profile versus elapsed time was plotted. Each confocal image from (A) and (C) is depicted by a cyan square. When returning to the 1.8 mM Ca2+ in the external solution, a secondary increase in fluorescence was detected, indicating SOCE activation and Ca2+ influx via activated Orai1 channels in the sarcolemma. (E) Distribution of the ratios of the peak of the slow Ca2+ transient (P2), highlighting the activation of SOCE, and that of the depleting-cocktail-induced Ca2+ transient (P1). When preincubated with 1 μM nifedipine (L-type Ca2+-channel blocker), the Ca2+ influx after store depletion remained unaltered, whereas in the presence of 15 μM BTP2 (a specific SOCE inhibitor), it was essentially eliminated in both strains. The number of cells examined is given in parentheses. (F) Representative Western blots illustrating the relative expression levels of STIM1 and Orai1, identified as the key proteins involved in SOCE. (G) In four independent experiments, the STIM1 and Orai1 endogenous protein level distributions were assessed as a percentage of the control. The asterisks in (E) and (G) denote a significant difference (p < 0.05) between WT and Cmpt; The hash mark in (E) denotes a significant difference (p < 0.001) between WT and BTP2/nifedipine-treated similar fibers. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

3. Figure 2
Unaltered release channel sensitivity to activation in fibers of Cmpt mice. Ca2+ transients were elicited under whole-cell voltage-clamp conditions by 100-ms-long progressively increasing membrane depolarizations ranging between −60 and +30 mV, with 10 mV increments every 500 ms in WT (A) and Cmpt (B) fibers. Each cell was held at −80 mV and perfused with 10 mM EGTA. Fluorescence was normalized to average resting F0(x). The white curves plot the spatially averaged F(t)/F0. (C) Data points represent the normalized maximal fluorescence at the given depolarization in six control (black) and seven Cmpt (red) muscle fibers. The voltage dependence of the normalized fluorescence was well fitted by a Boltzmann distribution. The mean values of parameters V1/2 and k were not significantly different: V1/2 (WT) = −26.03 ± 1.35 mV and k (WT) = 6.49 ± 1.15 mV, and V1/2 (Cmpt) = −28.86 ± 0.77 mV and k (Cmpt) = 6.91 ± 0.65 mV. (D) Normalized voltage dependence of the peak components of Ca2+-release flux. Values from five to six different experiments were averaged after normalization to the maximum of the individual Boltzmann fits. (E and F) Representative electron micrographs from WT and Cmpt FDB muscle samples, depicting the apparently unaltered calcium release units. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

4. Figure 3
Suppressed Ca2+ transients and release fluxes in Cmpt fibers. Line-scan images normalized to baseline fluorescence F0(x) in WT (A) and Cmpt (B) fibers subjected to a large, long-lasting (1 s) depolarization. The white curve represents the normalized fluorescence averaged over x. (C) [Ca2+]i(t), derived from ΔF(t)/F0 records in (A) (black) and (B) (red). (D) Release flux (Rrel) derived by the standard removal procedure from [Ca2+]i(t). Points of interest, peak (P), quasi-steady level (QS), and steady level (S) are indicated by dashed lines. (E) Net amount released, calculated by time integration of net release fluxes in (D). Details of the removal model parameters used in the calculation of flux are given in Materials and Methods and were identical for both cells. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

5. Figure 4
Increased fatigability of Cmpt muscles elicited by a train of pulses. The kinetic analysis of Ca2+-release fluxes for transients elicited by a train of depolarizing pulses to +30 mV at 1 Hz frequency, lasting 200 ms each in WT (A) and in Cmpt (B) FDB fibers. (C) Evolution of the release flux calculated from the white curves in (A) (black) and (B) (red). (D) Evolution of the Ca2+ amount released. (E) SR Ca2+ content estimated from the amount released minus the removal flux. A single-exponential function was fitted to the points (see Eq. A10 in Appendix A). (F) Pooled data of the calculated fluxes (K1) through Orai1 channels as predicted by our model (see Appendix A) shows a significant decrease in the mutant fibers. The asterisk denotes a significant difference (p < 0.05) between WT and Cmpt. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

6. Figure 5
Unaltered resting [Ca2+] but decreased SR Ca2+ content in the Cmpt muscles. (A) Resting [Ca2+]i was measured with fura-2AM and no differences were found between the two strains. (B) After application of 4-CmC, a potent RyR1 agonist, the SR Ca2+ content could be indirectly estimated, and significantly reduced values were found in the mutant. The number of cells studied is given in parentheses. The asterisk denotes a significant difference (p < 0.05) between WT and Cmpt fibers. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

7. Figure 6
Increased fatigability of Cmpt muscles elicited by a pulse train of short depolarizations. Line-scan images were recorded for WT (A) and Cmpt (B) fibers loaded with rhod-2 via the patch pipette and subjected to brief 20-ms-long depolarizing pulses to +30 mV at 3 Hz (50 consecutive pulses) to induce muscle fatigue. (C) The evolution of the release flux calculated from the white lines in (A) (black) and (B) (red). Note the smaller flux values for the mutant (red). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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8. Figure 7
A three-compartment model to estimate the time course of SR depletion. The model takes into account the release from and reuptake into the SR, as well as the influx through Orai channels and removal of calcium ions by the surface-membrane Ca2+ pump (see Appendix A). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions